Helper-free stocks of recombinant adeno-associated virus vectors

Abstract

A method for producing helper-free stocks of recombinant adeno-associated virus (rAAV) which can be used to efficiently and stably transduce foreign genes into host cells or organisms. The method comprises the cotransfection of eukaryotic cells with rAAV and with helper AAV DNA in the presence of helper virus (e.g. adenovirus or herpesvirus) such that the helper AAV DNA is not associated with virion formation. The crux of the invention lies in the inability of the helper AAV DNA to recombine with rAAV vector, thereby preventing the generation of wild-type virus. The pure stocks of recombinant AAV provide an AAV viral expression vector system with increased yield of recombinant virus, improved efficiency, higher definition, and greater safety than presently used systems.

1. A helper-free recombinant adeno-associated virus stock, said recombinant adeno-associated virus stock comprising a packaged recombinant adeno-associated virus vector containing a foreign DNA sequence, but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector, wherein said adeno-associated virus is produced by cells permissive for adeno-associated virus replication, said cells cotransfected with (i) a recombinant adeno-associated virus vector comprising a foreign DNA sequence but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector, and (ii) a recombinant helper adeno-associated virus DNA comprising adeno-associated virus genes required for replication or packaging, non-adeno-associated virus cis acting elements and lacking adeno-associated virus inverted terminal repeat sequences, wherein said cotransfected cells are infected with a helper virus.
 wherein said recombinant adeno-associated virus vector comprises up to 195 base pairs of the adeno-associated virus terminal repeated sequence, and shares no adeno-associated virus sequence with the helper adeno-associated virus DNA.  
 
     
     
       2. A helper-free recombinant adeno-associated virus stock, said recombinant adeno-associated virus stock comprising a packaged recombinant adeno-associated virus vector containing a foreign DNA sequence, but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector, wherein said adeno-associated virus is produced by an adeno-associated virus packaging cell line, transfected with a recombinant adeno-associated virus vector comprising a foreign DNA sequence but lacking adeno-associated virus genes required for replication or packaging of said adeno-associated virus vector and infected with a helper virus, wherein said recombinant adeno-associated virus vector comprises up to 195 base pairs of the adeno-associated virus terminal repeated sequence, and shares no adeno-associated virus sequence with the helper adeno-associated virus DNA. 
     
     
       3. A system for production of recombinant adeno-associated virus free of adeno-associated helper virus, comprising:
 (a) cells permissive for adeno-associated virus replication;  
 (b) a recombinant adeno-associated virus vector which contains a foreign DNA sequence which can be incorporated into an infectious virion and cis-acting sequences needed for DNA replication and packaging of said foreign DNA but lacking AAV DNA sequences encoding trans-acting products necessary for replication or packaging of said adeno-associated virus vector;  
 (c) a recombinant helper adeno-associated virus DNA sequence which provides viral functions sufficient for the replication and packaging into infectious virions of said recombinant adeno-associated virus vector, which lacks adeno-associated virus terminal repeat sequences and shares no adeno-associated virus sequences with said vector, and which cannot itself be incorporated into an infectious virion; and  
 (d) a helper virus.